Journal: Cancer Medicine

Article Title: MiR‐362 suppresses cervical cancer progression via directly targeting BAP31 and activating TGFβ/Smad pathway

doi: 10.1002/cam4.3601

Figure Lengend Snippet: MiR‐362 downregulates BAP31 expression by directly targeting BAP31 3'‐UTR. (A) Venn diagram depicting potential miRNA candidates that may regulate BAP31 expression in cervical cancer. (B) qRT‐PCR results of miRNA expression levels in normal and cervical cancer tissues, 12 cases in normal group and 28 cases in tumor group. (C) Immunoblotting analyses for BAP31 protein levels following transfection of seven miRNA mimics into HeLa and SiHa cells. (D) Results of luciferase reporter assays in HeLa and SiHa cells co‐transfected with BAP31 WT (wild type) 3′‐UTR vectors and seven miRNA mimics, respectively, for 48 h. (E) qRT‐PCR result of miR‐362 expression in HeLa and SiHa cell line. (F) Diagram of BAP31 3'‐UTR WT and MU vectors for the putative target sequence of miR‐362. WT: wild type; MU: mutant type. (G) qRT‐PCR analyses for miR‐362 level following transfection of miR‐362 mimics, NC (negative control), and siBAP (BAP31 siRNA) into HeLa cells, U6 RNA was used as a control. (H) Results of luciferase reporter assays in HeLa and SiHa cells co‐transfected with BAP31 WT/MU 3′‐UTR vectors and miRNA mimics for 48 h. (I) Immunoblotting analyses for BAP31 protein levels following transfection of NC, miR‐362 mimics, and siBAP into HeLa and SiHa cells. Transfection with BAP31 siRNA (siBAP31) acted as a positive control. * p < 0.05; ** p < 0.01; **** p < 0.0001. Data are represented as the mean ±SD of three independent experiments

Article Snippet: The antibodies used for immunoblotting are from CST (Cell Signaling Technology) for caspases, Smad pathway proteins, and SPTBN1, Abcam (Abcam) for BAP31 and cyclin family proteins, and Proteintech (Proteintech) for Actin.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Transfection, Luciferase, Sequencing, Mutagenesis, Negative Control, Control, Positive Control

Journal: Cancer Medicine

Article Title: MiR‐362 suppresses cervical cancer progression via directly targeting BAP31 and activating TGFβ/Smad pathway

doi: 10.1002/cam4.3601

Figure Lengend Snippet: Reduced miR‐362 levels in cervical cancer associated with BAP31 protein overexpression and patient survival. (A) MiR‐362 expression was determined via qRT‐PCR in 34 paracancer tissues and 219 cancer tissues, U6 RNA was used as a control. (B) Expressions of miR‐362 in different stages of cervical cancer, for 89 patients in stage Ⅰ, 71 patients in stage Ⅱ, and 59 patients in stage Ⅲ or Ⅳ. (C) Expressions of BAP31 protein in different stages of cervical cancer, getting from the histochemistry score results after immunohistochemistry. (D) Survival curves of five different cancers with low and high miR‐362 expression from TCGA database. * p < 0.05; **** p < 0.0001

Article Snippet: The antibodies used for immunoblotting are from CST (Cell Signaling Technology) for caspases, Smad pathway proteins, and SPTBN1, Abcam (Abcam) for BAP31 and cyclin family proteins, and Proteintech (Proteintech) for Actin.

Techniques: Over Expression, Expressing, Quantitative RT-PCR, Control, Immunohistochemistry

Journal: Cancer Medicine

Article Title: MiR‐362 suppresses cervical cancer progression via directly targeting BAP31 and activating TGFβ/Smad pathway

doi: 10.1002/cam4.3601

Figure Lengend Snippet: MiR‐362 regulates the proliferation and apoptosis of cervical cancer cells by targeting BAP31. (A) Apoptosis was analyzed by flow cytometry at 48 h post transfection with NC, miR‐362 mimics, miR‐362 mimics+BAP31 plasmid, and BAP31 siRNA. NC group acted as a NC and siBAP group as positive control. (B) Automated western immunoblotting analyses for caspase 3 and caspase 6 protein levels following transfection of NC, miR‐362 mimics, miR‐362 mimics+BAP31 plasmid, and BAP31 siRNA into HeLa cells. (C) The real‐time cell counting after transfection in the three groups. * showed the p value between mimics versus NC group. (D) Immunoblotting analyses for cyclin family protein levels following transfection in HeLa cells. (E) Flow cytometric analysis of the HeLa and SiHa cell cycle distributions with PI at 48 h post transfection with NC, mimics, miR‐362 mimics+BAP31 plasmid and siBAP. * p < 0.05; ** p < 0.01; *** p < 0.001. Data are represented as the mean ± SD of three independent experiments

Article Snippet: The antibodies used for immunoblotting are from CST (Cell Signaling Technology) for caspases, Smad pathway proteins, and SPTBN1, Abcam (Abcam) for BAP31 and cyclin family proteins, and Proteintech (Proteintech) for Actin.

Techniques: Flow Cytometry, Transfection, Plasmid Preparation, Positive Control, Western Blot, Cell Counting

Journal: Cancer Medicine

Article Title: MiR‐362 suppresses cervical cancer progression via directly targeting BAP31 and activating TGFβ/Smad pathway

doi: 10.1002/cam4.3601

Figure Lengend Snippet: MiR‐362 delays cervical cancer progression in vivo. (A) Implementation program of tumor‐bearing nude mice experiments. (B) Image of tumors after 3 weeks’ transfection. (C) Tumor volumes were measured after 3 weeks’ transfection. (D) Growth curve of xenografts of HeLa cells transfected with NC, mimics and siBAP. * showed the p value between mimics versus NC. (E) Kaplan–Meier means curve representing the overall survival of the injected mice. (F and G) H‐scores (histochemistry scores) and images (20×) of BAP31 immunohistochemical staining in tumor tissues from xenograft mouse model. * p < 0.05; ** p < 0.01. Data are represented as the mean ± SD of at least three independent experiments

Article Snippet: The antibodies used for immunoblotting are from CST (Cell Signaling Technology) for caspases, Smad pathway proteins, and SPTBN1, Abcam (Abcam) for BAP31 and cyclin family proteins, and Proteintech (Proteintech) for Actin.

Techniques: In Vivo, Transfection, Injection, Immunohistochemical staining, Staining

Journal: Cancer Medicine

Article Title: MiR‐362 suppresses cervical cancer progression via directly targeting BAP31 and activating TGFβ/Smad pathway

doi: 10.1002/cam4.3601

Figure Lengend Snippet: BAP31/SPTBN1 complex regulates cervical cancer cell behavior via TGFβ/Smad pathway. (A) Differentially expressed genes were identified in the HeLa wild‐type and knockout BAP31 cell by gene microarray. (B) KEGG pathway analyses of the gene chip using the IPA commercially available software. (C) Automated western immunoblotting for Smad family protein levels following transfection of NC, mimics, miR‐362 mimics+BAP31 plasmid, and siBAP into HeLa cells. (D) Genes were ranked according to their degree of correlation with BAP31 after co‐IP and MS analysis. (E) Immunoblotting analyses for SPTBN1 and BAP31 protein levels after transfection in HeLa cells. (F) Automated western immunoblotting for SPTBN1 and BAP31 protein after co‐IP with each other in HeLa cells. (G) Immunoblotting for SPTBN1, Smad 3, and BAP31 after co‐IP. (H) Immunofluorescence staining of HeLa cells with anti‐BAP31 and anti‐SPTBN1

Article Snippet: The antibodies used for immunoblotting are from CST (Cell Signaling Technology) for caspases, Smad pathway proteins, and SPTBN1, Abcam (Abcam) for BAP31 and cyclin family proteins, and Proteintech (Proteintech) for Actin.

Techniques: Knock-Out, Microarray, Software, Western Blot, Transfection, Plasmid Preparation, Co-Immunoprecipitation Assay, Immunofluorescence, Staining

Journal: Cancer Medicine

Article Title: MiR‐362 suppresses cervical cancer progression via directly targeting BAP31 and activating TGFβ/Smad pathway

doi: 10.1002/cam4.3601

Figure Lengend Snippet: Schematic diagram of the role of miR‐362 in cervical cancer. As a negative transcriptional regulator of BAP31, abnormal low expression of miR‐362 led to increased expression of BAP31 in CC. The combination of BAP31 and SPTBN1 blocked signal transduction, and TGFβ/Smad pathway was inhibited. As a result, cervical cancer cell proliferation is accelerated, and apoptosis is reduced. In contrast, cervical cancer cells overexpressing miR‐362 via treating with miR‐362 mimics exhibited a low level of BAP31 expression. Thus, SPTBN1 could bind to Smad 3, so that TGFβ/Smad pathway is activated to inhibit cell growth

Article Snippet: The antibodies used for immunoblotting are from CST (Cell Signaling Technology) for caspases, Smad pathway proteins, and SPTBN1, Abcam (Abcam) for BAP31 and cyclin family proteins, and Proteintech (Proteintech) for Actin.

Techniques: Expressing, Transduction

Journal: Virus Research

Article Title: Imidazonaphthyridine effects on Chikungunya virus replication: Antiviral activity by dependent and independent of interferon type 1 pathways

doi: 10.1016/j.virusres.2022.199029

Figure Lengend Snippet: RO8191 activity on CHKV -nanoluc replication. A) Representative structure of CHIKV- nanoluc genome [PDB: 6NK5]( Basore et al., 2019 ; Metz and Pijlman, 2016 ). B) Imidazonaphthyridine chemical structure (RO4948191 or RO8191) (CAS No.: 691,868–88–9; https://www.medchemexpress.com/ro8191.html ). C) Representative scheme of the infection assays. BHK-21 cells were treated with two-fold serial dilutions of RO8191 at concentrations ranging from 0.46 to 60 µM. CHIKV- nanoluc replication was quantified by measuring nanoluciferase activity (indicated by a black square) and cell viability using MTT assay (indicated by a gray circle). Mean ± SD values from a minimum of three independent experiments, each measured in triplicate, are represented. All images were generated using GraphPad Prism 8 and GIMP 2.1v.

Article Snippet: B) Imidazonaphthyridine chemical structure (RO4948191 or RO8191) (CAS No.: 691,868–88–9; https://www.medchemexpress.com/ro8191.html ).

Techniques: Activity Assay, Infection, MTT Assay, Generated

Journal: Virus Research

Article Title: Imidazonaphthyridine effects on Chikungunya virus replication: Antiviral activity by dependent and independent of interferon type 1 pathways

doi: 10.1016/j.virusres.2022.199029

Figure Lengend Snippet: RO8191 activity on different stages of CHIKV- nanoluc replicative cycle in BHK-21 cells. A) BHK-21 cells were treated with RO8191 for 1 h, cells were washed with PBS to remove the compound, and were infected with CHIKV- nanoluc at MOI 0.1 for 1 h. Then, the medium was removed, cells were washed to remove unbound virus, and fresh medium was added. B) BHK-21 cells were infected with CHIKV- nanoluc (MOI 0.1) and simultaneously treated with RO8191 for 1 h. Then, cells were washed to completely remove the inoculum, and a fresh medium was added. C) RO8191 and CHIKV- nanoluc at MOI 5 were incubated for 1 h (representative inoculum). Then, the inoculum was added to the cells for an additional hour. Cells were washed to remove the inoculum and fresh medium was added. D) BHK-21 cells were infected with CHIKV- nanoluc (MOI 0.1) for 1 h, cells were washed with PBS to remove unbound virus, and were treated with RO8191. All infection assays were quantified 16 h.p.i through the measurement of luminescence levels. Schematic representation of each time-based assay as indicated by BHK-21 cells (black bars), RO8191 (gray bars), CHIKV- nanoluc (orange bars), and CHIKV-RO8191 inoculum (blue tube). E) Schematic representation of the time-based BHK-CHIKV cell assay as indicated by the BHK-CHIKV-NCT cells (black bars) and RO8191 (gray bars). BHK-CHIKV-NCT cells were incubated for 72 h with the RO8191 compound at concentrations ranging from 60 to 0.46 µM. Then, cells were washed with PBS and lysed with Passive Lysis Buffer to measure the activity of Renilla . The reduction of Renilla activity in BHK-CHIKV-NCT cells is shown by 50% of the highest concentrations (EC 50 ) of RO8191 (black squares) and an effect in cell viability (CC 50 ) using MTT assay (gray circles). Mean ± SD values of a minimum of three independent experiments, each measured in triplicate. (***) P <0.001, (**) P <0.01, and (*) P <0.05 are represented. Images were generated using GraphPad Prism 8 and GIMP 2.1v.

Article Snippet: B) Imidazonaphthyridine chemical structure (RO4948191 or RO8191) (CAS No.: 691,868–88–9; https://www.medchemexpress.com/ro8191.html ).

Techniques: Activity Assay, Infection, Virus, Incubation, Lysis, MTT Assay, Generated

Journal: Virus Research

Article Title: Imidazonaphthyridine effects on Chikungunya virus replication: Antiviral activity by dependent and independent of interferon type 1 pathways

doi: 10.1016/j.virusres.2022.199029

Figure Lengend Snippet: BHK-21 cells express type-1 IFN (α/β) during RO8191 treatment. A) Percentage of CHIKV WT infection by measuring mRNA-CHIKV WT in supernatant of BHK-21 cells. B) CHIKV WT RNA titers by measuring mRNA-CHIKV WT in supernatant of BHK-21 cells. C) Percentage of CHIKV WT infection by measuring mRNA-CHIKV WT per mL in cell lysates of BHK-21 cells. D) CHIKV WT RNA titers by measuring mRNA-CHIKV WT per mL detected in cell lysates of BHK-21 cells. The CHIKV infection is presented in black bars while CHIKV in the presence of RO8191 treatment is demonstrated in gray bars. E) mRNA means of IFN-α expressed by BHK-21 during infection and/or treatment with RO8191. F) mRNA mean of IFN-β expressed by BHK-21 during infection and/or treatment with RO8191. BHK-21 cells were infected with CHIKV WT (MOI 0.1 PFU/well) in the presence or absence of RO8191 at 5 µM for 16 h. The supernatant and cell lysates were collected and processed to RNA extraction. cDNA was produced and mRNA levels was quantified by RT-qPCR. Mean values of three independent experiment each measured in triplicate, are represented. (****) P <0.0001. Images were generated using GraphPad Prism 8 and GIMP 2.1v.

Article Snippet: B) Imidazonaphthyridine chemical structure (RO4948191 or RO8191) (CAS No.: 691,868–88–9; https://www.medchemexpress.com/ro8191.html ).

Techniques: Infection, RNA Extraction, Produced, Quantitative RT-PCR, Generated

Journal: Virus Research

Article Title: Imidazonaphthyridine effects on Chikungunya virus replication: Antiviral activity by dependent and independent of interferon type 1 pathways

doi: 10.1016/j.virusres.2022.199029

Figure Lengend Snippet: RO8191 activity against CHIKV- nanoluc infection in Vero-E6 cells. A) Representative scheme of the infection assays. Vero-E6 cells were treated with three-fold serial dilutions of RO8191 at concentrations ranging from 0.02 to 60 µM. CHIKV replication was quantified by measuring nanoluciferase activity (indicated by a black square) and cell viability using an MTT assay (indicated by a gray circle). Values of EC 50 , CC 50, and SI were obtained from the treatment of Vero-E6 cells treated with RO8191. B) Vero-E6 cells were treated with RO8191 for 1 h, then the cells were washed with PBS and infected with CHIKV- nanoluc virus at MOI 0.1 for 1 h. The medium was removed, cells were washed with PBS and fresh medium was added. C) Vero-E6 cells were infected with CHIKV- nanoluc (MOI 0.1) and simultaneously treated with RO8191 for 1 h. Then, cells were washed, and fresh medium was added. D) RO891 and CHIKV- nanoluc at MOI 5 were incubated for 1 h, then the inoculum was added to cells for an extra 1 h, cells were washed, and fresh medium was added. E) Vero-E6 cells were infected with CHIKV- nanoluc (MOI 0.1) for 1 h, cells were washed with PBS and treated with RO8191. Schematic representation of each time-based assay as indicated by Vero-E6 cells (blue bars), RO8191 (gray bars), CHIKV- nanoluc (orange bars), and CHIKV- nanoluc /RO8191 inoculum (blue tube). Mean ± SD values of a minimum of three independent experiments, each measured in triplicate, are represented. (****) P <0.0001, (***) P <0.001 and (*) P <0.05. Images were generated using GraphPad Prism 8 and GIMP 2.1v.

Article Snippet: B) Imidazonaphthyridine chemical structure (RO4948191 or RO8191) (CAS No.: 691,868–88–9; https://www.medchemexpress.com/ro8191.html ).

Techniques: Activity Assay, Infection, MTT Assay, Virus, Incubation, Generated

Journal: Virus Research

Article Title: Imidazonaphthyridine effects on Chikungunya virus replication: Antiviral activity by dependent and independent of interferon type 1 pathways

doi: 10.1016/j.virusres.2022.199029

Figure Lengend Snippet: Localization and ChemPLP values showed between RO8191 and CHIKV proteins by molecular docking calculations.

Article Snippet: B) Imidazonaphthyridine chemical structure (RO4948191 or RO8191) (CAS No.: 691,868–88–9; https://www.medchemexpress.com/ro8191.html ).

Techniques:

Journal: Virus Research

Article Title: Imidazonaphthyridine effects on Chikungunya virus replication: Antiviral activity by dependent and independent of interferon type 1 pathways

doi: 10.1016/j.virusres.2022.199029

Figure Lengend Snippet: 2D and 3D interactions between RO8191 and CHIKV proteins showed by molecular docking analysis. A) RO8181 interacts with CHIKV glycoproteins mainly through Hydrogen Bond (green lines) and alkyl ligands (pink lines). B) RO8191 showed interactions with nsP1 protein mainly through Conventional Hydrogen Bonds (green circles) interactions. C) RO8191 interacts with nsP2 mainly through Alkyl (pink lines) and Hydrogen Bonds (green lines) interactions. D) RO8191 interacts with nsP3 mainly through Hydrogens Bonds ligands and interactions (green lines and circles). E) RO8191 interactions with nsP4 mainly through Conventional Hydrogens Bonds (green lines) and Halogen (blue lines) ligands. RO8191 (PDB: 3R0L) and the CHIKV proteins (glycoprotein complex [PDBid: 3N42], nsP1 [PDBid: 7DOP], nsP2 [PDBid:4ZTB], nsP3 [PDBid:6W8Z], and database representative sequence of the nsP4 extracted from the virus polyprotein [uniport-id: Q8JUX6]) were docked employing the GOLD program site using a genetic algorithm (GA) and the score ChemPL. Images were generated using GraphPad Prism 8 and GIMP 2.1v.

Article Snippet: B) Imidazonaphthyridine chemical structure (RO4948191 or RO8191) (CAS No.: 691,868–88–9; https://www.medchemexpress.com/ro8191.html ).

Techniques: Sequencing, Virus, Generated

Journal: Virus Research

Article Title: Imidazonaphthyridine effects on Chikungunya virus replication: Antiviral activity by dependent and independent of interferon type 1 pathways

doi: 10.1016/j.virusres.2022.199029

Figure Lengend Snippet: Infrared spectroscopy indicates molecular interactions between CHIKV virions with RO8191. A) Representative scheme of the ATR-FTIR technology with CHIKV virions (black line), RO8191 (blue line), and CHIKV virion plus incubation with RO8191 (red line). B) The representative infrared average spectrum of normalized mean spectra of CHIKV virion (black line), RO8191 (blue line), and CHIKV virion plus RO8191 (red line) employing ATR-FTIR platform from 1800 to 800 cm −1 . C-F) The representative mean spectra of second derivative analysis to the vibrational modes at 1496 cm −1 ( C ), 1465 cm −1 ( D ), 1448 cm −1 ( E ), 1333 cm −1 ( F ), and 1078 cm −1 ( G ). Images were generated using GraphPad Prism 8 and GIMP 2.1v.

Article Snippet: B) Imidazonaphthyridine chemical structure (RO4948191 or RO8191) (CAS No.: 691,868–88–9; https://www.medchemexpress.com/ro8191.html ).

Techniques: Spectroscopy, Incubation, Generated

Journal: Discover. Oncology

Article Title: Ritanserin suppresses acute myeloid leukemia by inhibiting DGKα to downregulate phospholipase D and the Jak-Stat/MAPK pathway

doi: 10.1007/s12672-023-00737-9

Figure Lengend Snippet: Agonists of Jak-Stat/MAPK signaling rescued the ritanserin-mediated anticancer effect and activates underlying pathways. The histograms of cell viability and apoptosis cell ratio after ritanserin combined with A the MAPK, MEK/ERK agonist C16-PAF or B the JAK/STAT agonists RO8191 for 48 h. Data are presented as the mean ± S.D. of triplicate experiments. (*p < 0.05; ***p < 0.001.) C The protein expression levels of p-MEK1/2(Ser217/221), MEK1/2, p-ERK1/2(Thr202/Tyr204) and ERK1/2 were assessed after treatment with ritanserin, C16-PAF or both for 48 h. D The protein expression levels of JAK1, JAK3, p-STAT5(Typ694), STAT5, p-STAT3(Tyr705) and STAT3 were assessed after treatment with ritanserin, RO8191 or both for 48 h

Article Snippet: The protein agonists applied were as follows: SphK1 agonist K6PC-5(HY-124042, MedChemExpress); MEK/ERK agonists C16-PAF (HY-108635, MedChemExpress); JAK/STAT agonists RO8191 (T22142, TargetMol).

Techniques: Expressing